HPV16 Phylogenetic Variants in Anogenital and Head and Neck Cancers: State of the Art and Perspectives.

HPV16 is responsible for approximately 60% and 90% of global HPV-induced cervical and oropharyngeal cancers, respectively. HPV16 intratype variants have been identified by HPV genome sequencing and classified into four phylogenetic lineages (A-D). Our understanding of HPV16 variants mostly derives from epidemiological studies on cervical cancer (CC) in which HPV16 B, C, and D lineages (previously named "non-European" variants) were mainly associated with high-grade cervical lesions and cancer. Although a predominance of HPV16 lineage A (previously named "European variants") has been observed in head and neck squamous cell carcinoma (HNSCC), epidemiological and in vitro biological studies are still limited for this tumor site. Next Generation Sequencing (NGS) of the entire HPV genome has deepened our knowledge of the prevalence and distribution of HPV variants in CC and HNSCC. Research on cervical cancer has shown that certain HPV16 sublineages, such as D2, D3, A3, and A4, are associated with an increased risk of cervical cancer, and sublineages A4, D2, and D3 are linked to a higher risk of developing adenocarcinomas. Additionally, lineage C and sublineages D2 or D3 of HPV16 show an elevated risk of developing premalignant cervical lesions. However, it is still crucial to conduct large-scale studies on HPV16 variants in different HPV-related tumor sites to deeply evaluate their association with disease development and outcomes. This review discusses the current knowledge and updates on HPV16 phylogenetic variants distribution in HPV-driven anogenital and head and neck cancers.

Prevailing of HPV-16 and 52 genotype in 2022-2023 in Sanandaj, Iran.

INTRODUCTION: Human papillomavirus (HPV) presents a potential threat to the onset of carcinogenesis in the cervix, anogenital regions, and oropharynx. HPV encompasses over 200 types, with at least 12 having the potential to cause cancer, impacting the majority of sexually active individuals. In this current research, we explore the occurrence and spread of HPV genotypes. MATERIAL AND METHODS: During this cross-sectional study conducted in Sanandaj, Iran from Feb 2022 to Aug 2023, diverse samples including oral, vaginal, and genital were collected from individuals referred to private laboratories in Sanandaj, Iran. After sample collection and DNA extraction (FAVORGEN, Taiwan), they were subjected to PCR and genotyping (MehrViru, Iran). The subsequent statistical analysis unveiled infection rates across different demographics and age groups. STATA (version 17) were used for statistical analysis. We examined infection rates across demographics using t-tests and Odds Ratio. RESULTS: Overall, 26% (249) out of 950 cases tested positive for HPV, with 69% of these classified as high-risk. Among the examined population, 98% (933) were female, and 2% (17) were male. Females aged 31-40 exhibited the highest percentage of HPV prevalence (115/460) in the study with the majority of positive cases belonging to HR genotypes. The overall most frequent genotypes identified were 6, 16, 52, 53, 51, 58, and 56. HPV-16 exhibited the highest frequency among HR genotypes, accounting for 42 (17%) occurrences, followed by HPV-52 with a frequency of 32 (13%). CONCLUSION: Our findings emphasize the significant prevalence of HPV among females, particularly in the 21-30 age group. The identification of high-risk genotypes, underscores the importance of targeted interventions for specific age cohorts. The age-stratified analysis highlights a consistent predominance of high-risk HPV across age groups, indicating the need for age-specific preventive measures. These results contribute valuable information for designing effective screening and vaccination strategies, to alleviate the impact of diseases associated with HPV.

Prognostic significance of human papillomavirus 16 viral load level in patients with oropharyngeal cancer.

Human papillomavirus (HPV) infection in patients with oropharyngeal squamous cell carcinoma (OPSCC) is a major determinant for better prognosis. However, there remain HPV-positive patients who have poor outcomes. The stratification strategy for detecting high-risk patients among those with HPV-positive OPSCC has not been well delineated, especially for Asian patients. We undertook a retrospective cohort study on the survival rate of 89 Japanese patients diagnosed with primary OPSCC. The tumors were concurrently analyzed for the presence of HPV E6 DNA/mRNA, viral DNA load, p16 expression, viral physical status, and viral variant lineage. Human papillomavirus 16 viral DNA was found in 45 (51%) OPSCCs. Human papillomavirus 16 DNA-positive OPSCCs with higher viral load (classified as HPV16 DNA-medium/high OPSCCs) showed significantly favorable overall survival and progression-free survival compared with HPV16 DNA-positive OPSCCs with lower viral load (<10 copies/cell; HPV16 DNA-low OPSCCs) and HPV16 DNA-negative OPSCCs. E6 mRNA expression was observed in all HPV16 DNA-medium/high OPSCCs but not in HPV16 DNA-low OPSCCs. Notably, p16-positive and HPV16 DNA-negative/low OPSCCs showed significantly worse survival than p16-positive and HPV16 DNA-medium/high OPSCCs and resembled HPV-unrelated OPSCCs with regard to survival and risk factor profile. Although not significant, a trend toward shorter survival was observed for HPV16-integrated OPSCCs. Phylogenetic analysis revealed two major types of HPV16 variants termed Asian (A4) and European (A1/A2/A3) variants, but no difference in survival between these variants was observed. Altogether, these findings suggest that HPV viral load is a potentially informative factor for more accurate risk stratification of patients with OPSCC.

Sequence variation analysis of the E1 and E2 genes of human papillomavirus type 16 in cervical lesions in women from the south of Poland.

The E1 and E2 genes of the human papillomavirus encode the so-called early proteins, their sequences are conserved, and regulatory functions are associated with the viral oncoproteins. The purpose of this study is to determine the HPV16 E1 and E2 mutations appearing in the female population of southern Poland, depending on the severity of cervical pathological changes. We also take into account the number of E1 and E2 mutations detected in the E6 gene variant (350G or 350T). This publication is one of the first in the Central and Eastern Europe to deal with this topic. We identified 4 mutations in the E1 gene and 24 mutations in the E2 gene that have not been described so far. In three cases of squamous cell carcinoma a C3409T mutation occurred, which is widely described as oncogenic. This mutation lies in the 3243-3539 area of the E2 hinge region. Statistical analyses show a possible relationship of mutations in this area with oncogenesis. The discovered dependencies may be important in the context of oncogenesis, however, a study with a larger group of patients is needed in order to confirm this view.

Genetic variants of HPV-16 and their geographical and anatomical distribution in men: A systematic review with meta-analysis.

BACKGROUND: The prevalence of Human Papillomavirus type 16 (HPV-16) variants in men and the association with tumor development has not been fully investigated. We estimated the prevalence of genital, anal, and oral HPV-16 infections in men through a systematic review and meta-analysis. METHODS: Seven databases were searched and included studies that identified HPV-16 positive males, HPV-16 variants (lineages/sublineages), and indicated the sample's anatomical origin. This protocol is registered in PROSPERO (CRD42020178013). RESULTS: The database searches yielded 14 studies including 445 HPV-16 positive samples classified as lineage A (n = 390), lineage D (n = 43), lineage B (n = 10), and lineage C (n = 2) variants. Lineage A variants predominated among the anatomical sites and the diverse geographical regions. CONCLUSIONS: HPV-16 lineages vary according to anatomical and geographical region. According to this preliminary evaluation of the current literature, we hypothesize that, similar to women, specific HPV-16 variants may also be associated to increased cancer risk in men.

The frequency of high-risk human papillomavirus types, HPV16 lineages, and their relationship with p16INK4a and NF-κB expression in head and neck squamous cell carcinomas in Southwestern Iran.

High-risk human papillomaviruses (hr-HPVs) are the key risk factors implicated in the development of a significant proportion of head and neck squamous cell carcinomas (HNSCCs). We aimed to investigate the distribution of hr-HPV types and HPV16 lineages in a sample of patients with HNSCC and the possible association between HPV status and the expression of P16INK4A and NF-κB in Iranian HNSCC patients. We examined 108 formalin-fixed, paraffin-embedded (FFPE) histologically confirmed primary SCC tissue specimens of different head and neck anatomical sites. HPV types and HPV16 lineages were determined by nested PCR and overlapping nested PCR assays, respectively, followed by gene sequencing and phylogenetic analysis. The expression of p16INK4a and NF-κB was evaluated by immunohistochemistry. Twenty-five (23.1%) HNSCC tissue specimens were tested positive for HPV infection. The most prevalent HPV type was HPV-16, followed by HPV18 and HPV11. HPV16 variants belonged to the lineage A and lineage D which were further sorted into sublineages A1, A2, and D2. A significant association between HPV status and p16INK4a immunoreactivity was observed in more than 76% of the HPV-related HNSCCs (P < 0.0001). The overexpression of p16INK4a and cytoplasmic NF-κB was more common in low-grade HNSCC tumors. Our data highlights that HPV16, in particular the A2 sublineage, followed by A1 and D2 sublineages are the major agents associated with HNSCCs in Iran. Based on HPV16 predominance and its lineage distribution pattern, it seems that the prophylactic vaccines developed for cervical cancer prevention could also be applicable for the prevention of HPV-related HNSCCs in our population.

Genetic signatures for lineage/sublineage classification of HPV16, 18, 52 and 58 variants.

Increasing evidences indicate that high-risk HPV variants are heterogeneous in carcinogenicity and ethnic dispersion. In this work, we identified genetic signatures for convenient determination of lineage/sublineage of HPV16, 18, 52 and 58 variants. Using publicly available genomes, we found that E2 of HPV16, L2 of HPV18, L1 and LCR of HPV52, and L2, LCR and E1 of HPV58 contain the proper genetic signature for lineage/sublineage classification. Sets of hierarchical signature nucleotide positions were further confirmed for high accuracy (>95%) by classifying HPV genomes obtained from Chinese females, which included 117 HPV16 variants, 48 HPV18 variants, 117 HPV52 variants and 89 HPV58 variants. The circulation of HPV variants posing higher cancer risk in Eastern China, such as HPV16 A4 and HPV58 A3, calls for continuous surveillance in this region. The marker genes and signature nucleotide positions may facilitate cost-effective diagnostic detections of HPV variants in clinical settings.

Virus-Host Protein-Protein Interactions between Human Papillomavirus 16 E6 A1 and D2/D3 Sub-Lineages: Variances and Similarities.

High-risk strains of human papillomavirus are causative agents for cervical and other mucosal cancers, with type 16 being the most frequent. Compared to the European Prototype (EP; A1), the Asian-American (AA; D2/D3) sub-lineage seems to have increased abilities to promote carcinogenesis. Here, we studied protein-protein interactions (PPIs) between host proteins and sub-lineages of the key transforming E6 protein. We transduced human keratinocyte with EP or AA E6 genes and co-immunoprecipitated E6 proteins along with interacting cellular proteins to detect virus-host binding partners. AAE6 and EPE6 may have unique PPIs with host cellular proteins, conferring gain or loss of function and resulting in varied abilities to promote carcinogenesis. Using liquid chromatography-mass spectrometry and stringent interactor selection criteria based on the number of peptides, we identified 25 candidates: 6 unique to AAE6 and EPE6, along with 13 E6 targets common to both. A novel approach based on pathway selection discovered 171 target proteins: 90 unique AAE6 and 61 unique EPE6 along with 20 common E6 targets. Interpretations were made using databases, such as UniProt, BioGRID, and Reactome. Detected E6 targets were differentially implicated in important hallmarks of cancer: deregulating Notch signaling, energetics and hypoxia, DNA replication and repair, and immune response.

Role of HPV 16 variants among cervical carcinoma samples from Northeastern Brazil.

BACKGROUND: Cervical cancer is the fourth most common type of cancer affecting women globally. In Brazil, it is the third most frequent type of cancer in women and HPV is present in approximately 90% of cases. Evidence suggests that variants of HPV 16 can interfere biologically and etiologically during the development of cervical cancer. METHODS: Cervix tumor fragments were collected, their DNA was extracted, and nested PCR was used to detect HPV. Positive samples were sequenced to determine the viral genotype. To characterize the HPV 16 strains, positive samples PCR was used to amplify the LCR and E6 regions of the HPV 16 virus. RESULTS: Data from 120 patients with cervical cancer were analyzed. Most women were between 41 and 54 years of age, had schooling until primary school, a family income between 1 and 2 times the minimum wage and were married/in a consensual union. There was no statistically significant association between HPV or socio-demographic variables and risk factors for cervical cancer (P <  0.05). HPV was present in 88 women (73%). The most prevalent types were HPV 16 (53.4%), HPV 18 (13.8%), HPV 35 (6.9%) and HPV 45 (5.7%). Of the 47 HPV 16 positive cases, variant A (49%) was present in 23 samples, followed by variant D in 20 cases (43%), and variants B and C in 2 cases each (4%). The most prevalent histological type of HPV 16 tumors was squamous cell carcinoma, followed by adenocarcinoma. There was a statistically significant association between HPV 16 variants and the tumors' histological types (P <  0.001). CONCLUSIONS: Knowledge of HPV 16 variants will provide data on their influence on the pathological and oncogenic aspects of cervical lesions.

Internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of human papillomavirus genotypes 16 and 18 using extracted DNA and samples treated with nucleic acid releasing agent.

Cervical cancer is primarily caused by persistent infection with high-risk human papillomavirus (HPV), and 70% of cases are associated with HPV16 and 18 infections. The objective of this study was to establish rapid, simple, and sensitive internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of HPV16 and 18. The assays were performed at 39 ℃ and were completed within 30 min. A total of 277 clinical samples of exfoliated cervical cells were tested by IC-RAA assays and commercial HPV real-time fluorescent PCR kits using extracted DNA and samples treated with nucleic acid releasing agent. The analytical sensitivity of the IC-RAA assay was found to be 10 copies/µL for the detection of HPV16 and 18 when using recombinant plasmids as targets. The optimal concentration of the internal control (IC) plasmid and 18 was 1000 copies/µL for HPV16 and 100 copies/µL for HPV18. The clinical sensitivity of the IC-RAA assays for HPV16 using extracted DNA and samples treated with nucleic acid releasing agent was 98.73% and 97.47%, respectively, with kappa values of 0.977 (P < 0.01) and 0.955 (P < 0.01), respectively, and 100% The specificity in both cases. For HPV18, the sensitivity and specificity were 100%, and the kappa value was 1 for both samples (P < 0.01). The IC-RAA assay is a promising tool for the detection of HPV16 and HPV18, especially in resource-constrained settings.

The D2 and D3 Sublineages of Human Papilloma Virus 16-Positive Cervical Cancer in Guatemala Differ in Integration Rate and Age of Diagnosis.

Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence. SIGNIFICANCE: This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

Prevalence and genotype distribution of human papillomavirus in Zhengzhou, China, in 2016.

In this study, the prevalence and genotype distribution of human papillomavirus (HPV) in 49,793 women aged 25-64 years were determined by fluorescent real-time polymerase chain reaction (PCR) assay. HPV was detected in 6,020 women, giving a prevalence of 12.09% (6020/49,793). Single and multiple infections accounted for 71.81% (4323/6020) and 28.19% (1697/6020) of total infections, respectively. The most commonly found genotypes were HPV52 (19.90%, 1198/6020) and HPV16 (19.17%, 1154/6020), followed by HPV58 (13.11%, 789/6020), HPV81 (10.10%, 608/6020) and HPV56 (9.00%, 542/6020). The prevalence of HPV increased with age and was highest in the 54- to 64-year-old age group. The genotypes covered by the nonavalent HPV vaccine accounted for 39.32% (2367/6020) and 22.81% (1373/6020) of the total monoinfections and polyinfections, respectively. This study indicates a high HPV infection rate in women in the city of Zhengzhou and a large percentage of women are infected with single or multiple high-risk HPV genotypes that cannot be prevented using the current nonavalent HPV vaccine. Vaccines incorporating more HPV genotypes and extended age coverage for the current nonavalent vaccine might be necessary to better prevent HPV-related cervical cancer.

Analysis of Human Papillomavirus (HPV) 16 Variants Associated with Cervical Infection in Italian Women.

This study aims to evaluate HPV16 variants distribution in a population of Italian women living in two different regions (Lombardy and Sardinia) by sequence analyses of HPV16-positive cervical samples, in order to reconstruct the phylogenetic relationship among variants to identify the currently circulating lineages. Analyses were conducted starting from DNA isolated from 67 HPV16-positive cervical samples collected from two different Italian centres (31 from Lombardy and 36 from Sardinia) of women with normal and abnormal cervical cytology. The entire long control region (LCR) and 300 nt of the E6 gene was sequenced to identify intra-type variants. Sequence comparison and phylogenetic analysis were made using a distance-based neighbour joining method (NJ) and Kimura two-parameter model. Data obtained reported that Italian sequences mainly belonged to the European lineage, in particular sublineage A2. Only five sequences clustered in non-European branches: two in North American lineage (sublineage D1), two in African-1 (sublineage B1) and one in African-2. A new 27 nucleotide duplication in the central segment of the LCR region was found in a sequence obtained from a sample isolated in Sardinia. A predominance of European variants was detected, with some degree of variability among the studied HPV16 strains. This study contributes to the implementation of data regarding the molecular epidemiology of HPV16 variants.

Correlation of Swede score colposcopy scoring system and histopathological results in patients with high-risk HPV infection other than HPV16 and 18.

OBJECTIVE: Triage with HPV genotyping has some caveats and debates for HPV positive cases other than 16 and 18. The Swede score colposcopic scoring system has not previously been evaluated in this group of patients. OBJECTIVE: To use the Swede score colposcopic scoring system to compare scores and final histopathological results in women who have undergone colposcopy owing to infection with high risk-HPVs other than HPV16 and 18 and to establish new cut-off values to predict pre-malignant lesions in this group of patients. METHODS: This study was conducted in 613 women undergoing colposcopic evaluation because of abnormal cervical cytology together with high-risk HPV infection. All patients referred were evaluated by an expert colposcopist, given a Swede score (using the Swede score colposcopic scoring system) by using five variables (acetowhiteness, margins plus surface, vessel pattern, lesion size, and iodine staining), and had at least one biopsy procedure (either colposcopically directed or by a loop electrical excision procedure). Sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratio values, and receiver operating characteristic curves for each clinico-pathological variable to detect low-grade and high-grade squamous intra-epithelial lesions, and any squamous cell abnormality (low-grade + high-grade squamous intra-epithelial lesions) were evaluated individually. RESULTS: Final histopathological results of the patients were normal in 53.2% of cases, low-grade lesions in 32.5% of cases, and high-grade lesions in 14.4% of cases. Swede score was ≥8 (median 7.97) for high-grade lesions and ≥5 (median 5.06) for low-grade lesions. The area under the curve values (95% CI) of Swede scores for low-grade and high-grade squamous intra-epithelial lesions, and low-grade + high grade lesions were 0.92, 0.98, and 0.96, respectively. A Swede score cut-off value ≥6 had a sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratios of 92%, 98%, 93%, 98%, and 50 (22.6 to 110.8), respectively, for high-grade lesions at the final pathology (P<0.001). One high-risk HPV type (except 16 and 18) was no better than another for calculating the median Swede score during colposcopy (P=0.43). CONCLUSIONS: The Swede score colposcopic scoring system appears to be a useful tool for evaluating atypical cervical cytology in women with high-risk HPV infection other than HPV types 16 and 18.

Human Papillomavirus (HPV) 16 and 18/45 Genotyping-Directed Follow-up of Women With Messenger RNA HPV-Positive, Cytology-Negative Cervical Screening Test Results.

OBJECTIVES: In this study, we sought to correlate genotype test results for human papillomavirus (HPV) types 16, 18, and 45 with histopathologic follow-up diagnoses in patients with messenger RNA (mRNA) high-risk HPV-positive, cytology-negative results. METHODS: We identified 1,157 patients with mRNA HPV-positive, cytology-negative cervical screening test results between June 2015 and June 2018. Reflex HPV 16/18/45 genotype results were documented in 1,018 women aged 30 years or older, 318 of whom had follow-up within 18 months. RESULTS: Histopathologic findings of cervical intraepithelial neoplasia 2 or worse (CIN2+) were diagnosed in 14 of 122 (11.5%) patients positive for HPV 16/18/45 vs in seven of 196 (3.6%) HPV 16/18/45-negative patients. Three patients with high-risk HPV-positive, cytology-negative cervical screening test results were diagnosed with stage I cervical adenocarcinomas following early colposcopic referral and biopsy after HPV 16/18/45-positive genotype results. CONCLUSIONS: Immediate reflex HPV 16/18/45 genotyping of mRNA HPV-positive, cytology-negative patients led to early colposcopic referral and histopathologic diagnoses of three difficult-to-detect, low-stage, cervical adenocarcinomas and significantly increased overall early detection of CIN2+ lesions.

HPV33+ HNSCC is associated with poor prognosis and has unique genomic and immunologic landscapes.

OBJECTIVE: To determine the influence of high-risk HPV genotype on outcomes in HNSCC patients. MATERIALS AND METHODS: This is a retrospective analysis of The Cancer Genome Atlas HNSCC cohort. RESULTS: Using multivariate Cox regression analysis, we revealed that HPV33+ HNSCC patients have inferior overall survival compared to HPV16+ HNSCC patients independent of anatomical site (HR 3.59, 95% CI 1.58-8.12; p = 0.002). A host anti-viral immune response, apolipoprotein B mRNA editing enzyme, and catalytic polypeptide-like mutational signature, was under represented and, aneuploidy and 3p loss were more frequent in HPV33+ tumors. A deconvolution RNA-Seq algorithm to infer immune cell fractions revealed that CD8+ cytotoxic T-cell infiltration was reduced in HPV33+ compared to HPV16+ tumors (1.3% vs. 2.7%, p = 0.007). TGFB1, a negative modulator of T-cell infiltration and function, showed expression and pathway enrichment in HPV33+ tumors. CONCLUSIONS: Our work reveals that HPV genotype, in particular HPV33, has a powerful impact on HNSCC patient survival. We argue that p16 immunohistochemistry as a surrogate biomarker for HPV+ status will lead to sub-optimal risk stratification and advocate HPV genotype testing as standard of care.

Evaluation of loop-mediated isothermal amplification assay for detection and typing of human papilloma virus 16 and 18 from endocervical samples.

Background: Many human papillomavirus (HPV) types are associated with cervical cancer (CC). Therefore, HPV genotyping has both clinical and epidemiological importance. HPV 16 and 18 are two principal high-risk types responsible for more than 70% of all CC cases. Although several commercial and non-commercial genotyping assays are available, there is a need for a cost-effective and sensitive genotyping method for low- and middle-income countries. Methods: The study was aimed at evaluation of loop-mediated isothermal amplification (LAMP) assay for HPV genotyping in cervical samples. A total of six primer sets for each HPV type were selected for the assay. The LAMP assay was standardised and validated with HPV control panel. Cervical biopsies were subjected to nested multiplex polymerase chain reaction (NM-PCR; as a part of routine diagnostic workup) and LAMP (HPV 16 and 18) simultaneously. Results: A total of 225 clinical samples were processed during the study period. The sensitivity of the assay was determined using the 10-fold dilutions of positive controls. Both the HPV 16-LAMP and HPV 18-LAMP assays were shown to detect as low as 10 viral copies per reaction, which is similar to that of NM-PCR. The LAMP assay had a good agreement (new cases; 92%, post-chemoradiotherapy [post-CRT]; 89.1%) with NM-PCR for the detection of both HPV 16 and 18. As compared to histology (new cases; 79.8%, post-CRT; 51.3%), LAMP had better agreement with NM-PCR for detection of HPV from post-CRT cases. Conclusions: We evaluated the LAMP assay for simultaneous detection and typing of HPV 16 and 18. The assay had good agreement with NM-PCR for detection of both HPV 16 and 18. The LAMP assay is a promising tool for HPV genotyping along with routine cervical cytology, especially in resource-constrained settings.

Predominance of high-risk human papillomavirus genotype 16 and 39 in women with premalignant and malignant cervical pathology from Raipur, Chhattisgarh: Clinical evaluation of tagging oligonucleotide cleavage and extension mediated genotyping assay.

Background: Identification of 14 high-risk human papillomavirus (HR-HPV) is immensely important in elucidating molecular epidemiology, patient monitoring and evidence-based treatment. There is paucity of such data from Chhattisgarh state of Central India. The present study has evaluated tagging oligonucleotide cleavage and extension-mediated Anyplex HR-HPV genotyping assay in identification of 14 HR-HPV genotypes attributable to premalignant and malignant cervical lesion in comparison to GP5+/6+ assay, cytology and colposcopy. Materials and Methods: A total of 185 clinically suspected cases of premalignant and malignant cervical lesion were investigated by HR-HPV genotyping, GP5+/6+, cytology and colposcopy. Results: Genotyping assay showed clinical sensitivity and specificity of 86.5% (confidence interval [CI]: 80.7-91.0) and 100% (CI: 86.3-100) respectively and found noninferior to GP5+/6+ assay (P > 0.05). HR-HPV prevalence was 76.3%, 88.4%, 94.8%, 100% and 100% among cervical intraepithelial neoplasia (CIN) Grade I-III, squamous cell carcinoma and adenocarcinoma cases, respectively. The four most common genotypes detected in CIN I-III were HPV 16 (63.9%), HPV 39 (15.0%), HPV 18 (6.0%) and HPV 33 (5.3%). In cervical cancer (CC) cases, HPV 16 (44.4%), HPV 39 (11.1%), dual infection of HPV 16, 18 (11.1%) and triple infection of HPV 16, 18, 33 (11.1%) were the four most identified genotypic aetiologies. A novel coinfection of HR-HPV 35, 39 were found in two and one cases of CIN I and II. Finding of HPV 39 as the second most prevalent genotype was unusual and underscores the importance of genotyping screening. Conclusion: Anyplex HR-HPV assay is arguably the useful assay for better patient management and can be useful for HR-HPV screening by its unique individual genotype identification of all HR-HPV. Finding of HPV 16, 39, 18, 33 and coinfection of 16,18 and 16, 18, 33 in CIN and CC would help vaccine manufacturer to design specific future HPV polyvalent vaccine preparation to curb down the CC-associated mortality.

HPV viral load in self-collected vaginal fluid samples as predictor for presence of cervical intraepithelial neoplasia.

OBJECTIVE: This study was performed to evaluate the use of high-risk HPV (hrHPV) viral load in screening tests for cervical cancer to predict persistent infection and presence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+). METHODS: We followed women between 30 and 60 years of age who performed self-sampling of vaginal fluid and subsequently a hrHPV test. Women who were hrHPV positive in their screening test repeated the hrHPV test 3-6 months later and were included in the present study. RESULTS: Our results show that women with a persistent HPV16 infection had higher HPV viral load in their primary screening test than women with transient infections (p = 5.33e-03). This was also true for sum of viral load for all hrHPV types in the primary screening test (p = 3.88e-07). 48% of women with persistent HPV16 infection and CIN2+ had an increase in HPV16 titer in the follow-up test, as compared to only 20% of women with persistent infection but without CIN2+ lesions. For the sum of all hrHPV types, 41% of women with persistent infection and CIN2+ had an increase in titer as compared to 26% of women without CIN2 + . CONCLUSIONS: The results show that hrHPV viral load in the primary screening HPV test is associated with the presence of CIN2+ and could be used in triaging hrHPV positive women for different follow-up strategies or recall times. Serial testing of hrHPV viral load has the potential to distinguish women with CIN2+ lesions from women with persistent infection but without CIN2+ lesions.

Mutational landscape and intra-host diversity of human papillomavirus type 16 long control region and E6 variants in cervical samples.

Human papillomavirus genotype 16 (HPV16) is the most frequent high-risk HPV (HR-HPV) identified in cervical precursor lesions and cervical cancer (CC) worldwide. The oncogenic potential of HPV16 is partly dependent on the lineage involved in the infection and the presence of clinically relevant mutations. In this report, we present the distribution of HR-HPV and the mutational profile and intra-host variability of HPV16 lineages, based on analysis of the long control region (LCR) and the E6 gene in samples with normal cytology (n = 39), squamous intraepithelial lesions (n = 25), and CC (n = 39). HR-HPV genotyping was performed using multiplex real-time PCR. HPV16 lineage assignments and mutation frequencies were determined by conventional PCR and Sanger DNA sequencing, and intra-patient viral populations were analyzed using next-generation sequencing (NGS). The most frequent HR-HPV type was HPV16, followed by HPV31 and HPV18. The frequency of HPV16 sublineages was A1/A2 > D2 > D3 and B1. Moreover, the most frequent mutations, both in samples from this study and in the available sequences from Mexican isolates in the GenBank database were LCR-G7518A, which is involved in carcinogenesis, and E6-T350G (producing L83V), associated with persistence of infection. Otherwise, deep sequencing revealed high conservation of viral lineages and mutations, independently of the stages studied. In conclusion, the high frequency and stability of these molecular markers, as well as the circulating viral lineages, could be related to the incidence of CC associated with HPV16. Hence, they deserve a broader analysis to determine the risk of specific populations for progression of the disease.